We previously reported that one major trifluoroacetylated (TFA) protein (TFA-59 kDa) was detected in rat liver microsomes 12 hr after halothane (H) treatment. Its CO-reduced difference spectrum (450.6 nm) was consistent with it being cytochrome P- 450. We have now further characterized this protein and indeed it appears to be a constitutive form of cytochrome P-450. When rat liver microsomes from untreated rats were incubated with H and NADPH, the 59 kDa protein became labeled with the TFA moiety, suggesting that it had metabolized H to the reactive acylating species, trifluoroacetyl halide. Antiserum prepared against TFA- 59 kDa reacted with proteins in the microsomes for liver (59 and 60 kDa), lung (59 kDa), adrenal (59 kDa), testes (59 kDa), and fat (59 kDa) of untreated rats and in the liver of a human (59 kDa). A 59-kDa cytochrome P-450 purified rabbit liver (JBC 260:2027, 1985), which hydroxylates regioselectively prostaglandins in the omega position, cross-reacted with the anti-TFA 59 kDa antiserum and also appeared to become labeled with the TFA group, when halothane was administered to rabbits. It appears that the 59 kDa protein is a previously uncharacterized form of rat cytochrome P-450, which is immunochemically related to rabbit and human liver proteins and several other proteins in various tissues of the body.